Artery- and vein-specific proteins and uses therefor

ABSTRACT

Arterial and venous endothelial cells are molecularly distinct from the earliest stages of angiogenesis. This distinction is revealed by expression on arterial cells of a transmembrane ligand, called EphrinB2 whose receptor EphB4 is expressed on venous cells. Targeted disruption of the EphrinB2 gene prevents the remodeling of veins from a capillary plexus into properly branched structures. Moreover, it also disrupts the remodeling of arteries, suggesting that reciprocal interactions between pre-specified arterial and venous endothelial cells are necessary for angiogenesis.

RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.09/085,820, filed May 28, 1998, which is a continuation-in-part of U.S.application Ser. No. 09/083,546, filed May 22, 1998. This applicationalso claims the benefit of U.S. Provisional Application No. 60/081,757,filed Apr. 13, 1998. The entire teachings of the above applications areincorporated herein by reference.

FUNDING

[0002] The invention was supported, in whole or in part, by the HowardHughes Medical Institute

BACKGROUND OF THE INVENTION

[0003] The process of blood vessel formation is fundamental in bothdevelopment and disease. The circulatory system is the first organsystem to emerge during embryogenesis, and is necessary to nourish thedeveloping fetus. Disorders of the circulatory system, such as coronaryartery disease, are a major cause of morbidity and mortality in modernsociety. Thus, repairing, replacing and promoting the growth of bloodvessels is a major target of clinical research and of pharmaceuticaldevelopment. Conversely, the ingrowth of new capillary networks intodeveloping tumors is essential for the progression of cancer. Thus, thedevelopment of drugs that inhibit this process of tumor angiogenesis isan equally important therapeutic goal. Little attention has been paid tothe problem of how arteries and veins acquire their distinct identities.Indeed, many people have assumed that the anatomical and functionaldifferences between arteries and veins simply reflect physiologicalinfluences, such as blood pressure, oxygenation and shear forces.Additional knowledge of how arteries and veins acquire their respectiveidentities would be valuable in both research and clinical settings.

SUMMARY OF THE INVENTION

[0004] The present invention relates to a method of distinguishingbetween arterial endothelial cells and venous endothelial cells based onthe expression of a protein on one type of such endothelial cells (e.g.,on arterial endothelial cells) and not on the other (e.g., not on venousendothelial cells) and to a wide variety of processes, methods andcompositions of matter, including those useful in research and clinicalsettings, which are based on the difference in expression between thetwo cells types. As described herein, it has been shown that there is amolecular distinction between arterial endothelial cells (arteries) andvenous endothelial cells (veins) and that arterial endothelial cells andvenous endothelial cells bear molecular markers which can be used toidentify, separate, target, manipulate or otherwise process each celltype specifically (separate from the other). As a result, arteries andveins can now be distinguished from one another, assessed for othergenetic molecular or functional differences and targeted, manipulated orotherwise processed individually or separately for research, diagnosticand therapeutic purposes.

[0005] The present invention relates to methods of distinguishing orseparating arterial endothelial cells (arteries) from venous endothelialcells (veins) based on their respective molecular markers; methods ofselectively targeting or delivering drugs or agents to arteries orveins; methods of altering (enhancing or inhibiting, where “inhibiting”includes partially or completely inhibiting) the function ofartery-specific or vein-specific molecular markers or interactionbetween them (and, thus, enhancing or inhibiting the effect suchfunctions or interactions have on arterial endothelial cells or venousendothelial cells); and methods of screening for drugs which actselectively on arterial endothelial cells or venous endothelial cells.The invention also relates to transgenic nonhuman mammals, such astransgenic mice, in which genes encoding an arterial endothelial cellmolecular marker or a venous endothelial cell molecular marker arealtered, either physically or functionally, and their use as “indicatormice” to specifically visualize either arteries of veins, to assess thefunction of the molecular marker which has been altered and to identifydrugs which affect (enhance or inhibit) their function. It furtherrelates to antibodies which bind an arterial endothelial cell-specificmarker or a venous endothelial cell-specific marker; viral or othervectors targeted to arteries or veins by virtue of their containing andexpressing, respectively, an arterial endothelial cell-specific markeror a venous endothelial cell-specific marker; cDNAs useful for preparinglibraries to be screened for additional artery- or vein- specific genesand immortalized cell lines derived from isolated arterial endothelialcells or venous endothelial cells or from transgenic animals (e.g.,mice) of the present invention.

[0006] A molecular marker for an arterial endothelial cell or a venousendothelial cell is any gene product (protein or RNA or combinationthereof) expressed by one of these cell types and not on the other. Suchmarkers are also referred to, respectively, to as arterial endothelialcell-specific (artery-specific) and venous endothelial cell-specific(vein-specific) products or proteins. In specific embodiments, they arereferred to, respectively, as arterial endothelial cell-specific(artery-specific) ligands and venous endothelial cell-specific(vein-specific receptors. Such molecular markers can be expressed oncell types in addition to arterial or venous endothelial cells, but arenot expressed on both arterial and venous endothelial cells. Molecularmarkers can include, for example, mRNAs, members of ligand-receptorpairs, and any other proteins such as adhesion proteins, transcriptionfactors or antigens which are not expressed on both cell types. In oneembodiment, the molecular marker is a membrane receptor which is thereceptor for a growth factor which acts on arteries or veins (e.g.,fibroblast growth factor-2 (FGF), vascular endothelial growth factors(VEGF 1-3, angiopoietins). In another embodiment the molecular marker isa member of an endothelial cell surface ligand-receptor pair which isexpressed on arterial or venous endothelial cells, but not on both. Forexample, as described in detail herein, a member of the Ephrin family ofligands and a member of the Eph family of receptors which is itsreceptor are molecular markers for arterial endothelial cells and venousendothelial cells, respectively and are useful to distinguish the twocell types. Any Ephrin family ligand which is expressed on arterialendothelial cells, but not on venous endothelial cells and a venousendothelial cell-specific Eph family receptor which binds the arterialendothelial cell-specific ligand can be used to distinguish betweenarteries and veins.

[0007] In one embodiment, the present invention relates to the discoverythat arterial endothelial cells express an Ephrin family ligand andvenous endothelial cells express an Eph family receptor which is areceptor of the Ephrin family ligand expressed on the arterialendothelial cells; methods of distinguishing or separating arterialendothelial cells (arteries) from venous endothelial cells (veins);methods of selectively targeting or delivering drugs or agents toarteries or veins; methods of enhancing or inhibiting angiogenesis, suchas by altering (increasing, decreasing or prolonging) activity of atleast one member of an Ephrin family ligand-cognate Eph family receptorpair and drugs useful in the methods; and methods of screening for drugswhich selectively act on arterial endothelial cells or venousendothelial cells. It further relates to transgenic nonhuman mammals,such as transgenic mice, which have altered genes encoding an Ephrinfamily ligand or altered genes encoding an Eph family receptor, such asEphrinB2 knockout mice which contain a tau-lacZ (tlacZ) insertion thatmarks arteries but not veins or EphB4 knockout mice which contain areporter construct (e.g., lacZ or alkaline phosphates gene) in the EphB4locus; methods of using these mice as “indicator mice” to define andvisualize angiogenic processes (e.g., tumor angiogenesis andischemia-associated cardiac neovascularization) or to screen drugs fortheir angiogenic or anti-angiogenic effects on arteries or veins invivo; and cells, such as immortalized cells, derived from the transgenicmice. The present invention also relates to antibodies which bind anartery-specific Ephrin family receptor (e.g., antibodies which bindEprhinB2); antibodies which bind a venous-specific Eph family receptor(e.g., antibodies which bind EphB4); viral or other vectors which aretargeted to arteries or veins for vessel-specific gene therapy by virtueof their containing and expressing DNA encoding, respectively, an Ephrinfamily ligand (e.g., EphrinB2) or an Eph family receptor (e.g., EphB4);cDNAs useful for preparing libraries to be screened for additionalartery-specific or vein-specific genes (whose gene products, in turn,might be artery-or vein-specific drug targets) and methods of repairingor replacing damaged arteries or veins by transplantation of isolatedarterial or venous endothelial cells, immortalized cell lines derivedfrom them or synthetic vessels configured from these cells.

[0008] As described herein and as is known to those of skill in the art,Ephrin family ligands are divided into two subclasses (EphrinA andEphrinB) and Eph family receptors are divided into two groups (EphA andEphB). As is also known, within each subclass or group, individualmembers are designated by an arabic number. The invention is describedherein with specific reference to EphrinB2 and EphB4, However, otherEphrin family ligand-Eph family receptor pairs which show similarartery-and vein-specific expression and their uses are also the subjectof this invention. Similar artery- and vein-specific pairs can beidentified by methods known to those of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1A is a diagram of the wild type locus of the EphrinB2 geneshowing the Exon-1 structure. The filled box represents 5′ untranslatedregion. The hatched box starts at the ATG, and includes the signalsequence. H=HindIII; X=XbaI; N=NcoI; E=EcoRI.

[0010]FIG. 1B is a diagram of the targeting vector used to disrupt theEphrinB2 gene.

[0011]FIG. 1C is a schematic representation of the mutated EphrinB2locus.

DETAILED DESCRIPTION OF THE INVENTION

[0012] As described herein, it has been shown that arteries and veinsare genetically distinct from the earliest stages of embryonicdevelopment and that reciprocal interactions between arteries and veinsare essential for proper vessel formation. This finding not only changesdramatically our view of the basic ontogenetic anatomy of embryonicvasculature, but also provides the means to distinguish between arterialendothelial cells and venous endothelial cells, both physically andfunctionally. As a result, means of separating the two cell types fromone another; of identifying other artery- or vein-specific genes; ofassessing the selective effects of drugs or other agents on arteries orveins and, thus, identifying those which are artery- or vein-specific;and of selectively delivering or targeting substances to either celltype are now available. In addition, the work described herein makes itpossible to modulate (enhance or inhibit) or control vasculogenesis andangiogenesis and to do so, if desired, in an artery- specific or vein-specific manner.

[0013] As described in the examples, a gene which encodes a cellmembrane-associated ligand which is present in the nervous system andthe vascular system has been shown to be expressed by arterialendothelial cells and not by venous endothelial cells. Further, the genewhich encodes the receptor for the ligands has been shown to beexpressed by venous endothelial cells, but not by arterial endothelialcells. Thus, for the first time, an arterial endothelialcell-(artery-)specific marker and a venous endothelialcell-(vein-)specific marker are available, making it possible todistinguish between arteries and veins for a variety of purposes, suchas further study and understanding of the mechanisms of blood vesselformation; selective targeting of treatments or therapies to arteries orveins (targeting to arteries but not veins or vice versa) and selectivemodulation (enhancement or inhibition) of formation, growth and survivalof arteries and/or veins.

[0014] In addition, the work presented in the examples demonstrates thatreciprocal signaling between arteries and veins is crucial for vesselmorphogenesis (development/formation of arteries and veins). Asdescribed, deletion of the ligand-encoding gene in mice prevented theproper development of both arterial and venous vessels. Since the ligandis present on arteries (but not veins), the occurrence of the venousdefect is evidence that veins require a signal from arteries for vesselmorphogenesis. Conversely, since the arteries are also defective in themutant mice, the ligand must have a function in the arterial cellsthemselves, in addition to its role in signaling to the veins. In viewof the fact the ligand present on arterial endothelial cells is atransmembrane structure, it most likely functions to receive andtransduce to arterial cells a reciprocal signal from venous cells.

[0015] Specifically, a ligand which is a member of the Ephrin family ofEph family receptor interactive proteins (Eph family of transmembraneligands) has been shown to be expressed by arterial endothelial cells,but not by venous endothelial cells. Thus, it is now possible todistinguish between or target arteries and veins by relying on thepresence or absence of an Ephrin family ligand and its receptor, whichis a member of the Eph family of receptor protein-tyrosine kinases. Asdescribed herein, arterial endothelial cells have been shown to expressEphrinB2 and venous endothelial cells have been shown to express EphB4,which is an EphrinB2 receptor. EphrinB2 is not expressed on venousendothelial cells and EphB4 is not expressed on arterial endothelialcells, providing a means by which the two cell types can be identifiedor distinguished and, thus, a means by which arterial endothelial cellsand venous arterial cells can be, for example, separated from oneanother, targeted specifically or acted upon in a selective manner(e.g., by a drug or agent which acts upon one cell type to the exclusionof the other).

[0016] The work described herein, particularly in the examples, refersto EphrinB2 and EphB4. However, any ligand-receptor pair from theEphrin/Eph family, any other ligand-receptor pair or any gene productproduced by one cell type and not the other (e.g., an Ephrin ligand isexpressed by arterial endothelial cells but not by venous endothelialcells and an Eph receptor is expressed by venous endothelial cells butnot by arterial endothelial cells) can be used to distinguish between oridentify and, thus, selectively act upon, arterial endothelial cells andvenous arterial cells.

[0017] The ephrins (ligands) are of two structural types, which can befurther subdivided on the basis of sequence relationships and,functionally, on the basis of the preferential binding they exhibit fortwo corresponding receptor subgroups. Structurally, there are two typesof ephrins: those which are membrane-anchored by aglycerophosphatidylinositol (GPI) linkage and those anchored through atransmembrane domain. Conventionally, the ligands are divided into theEphrin-A subclass, which are GPI-linked proteins which bindpreferentially to EphA receptors and the ephrinB subclass, which aretransmembrane proteins which generally bind preferentially to EphBreceptors.

[0018] The Eph family receptors are a family of receptorprotein-tyrosine kinases which are related to Eph, a receptor named forits expression in an erythropoietin-producing human hepatocellularcarcinoma cell line. They are divided into two subgroups on the basis ofthe relatedness of their extracellular domain sequences and theirability to bind preferentially to ephrinA proteins or ephrinB proteins.Receptors which interact preferentially with ephrinA proteins are EphAreceptors and those which interact preferentially with ephrinB proteinsare EphB receptors.

[0019] As used herein, the terminology Ephrin and Eph are used to refer,respectively, to ligands and receptors. They can be from any of avariety of animals (e.g., mammals/nonmammals,vertebrates/nonvertebrates, including humans). The nomenclature in thisarea has changed rapidly and the terminology used herein is thatproposed as a result of work by the Eph Nomenclature Committee, whichcan be accessed, along with previously-used names at web sitehttp://www.eph-nomenclature.com. For convenience, eph receptors andtheir respective ligand(s) are given in the Table. EPH RECEPTORS ANDLIGAND SPECIFICITIES Eph Receptors Ephrins EphA1 Ephrin-A1 EphA2Ephrin-A3, -A1, A5, -A4 EphA3 Ephrin-A5, -A2, A3, -A1 EphA4 Ephrin-A5,-A1, A3, -A2, -B2, -B3 EphA5 Ephrin-A5, -A1, A2, -A3, -A4 EphA6Ephrin-A2, -A1, A3, -A4, -A5 EphA7 Ephrin-A2, -A3, A1 EphA8 Ephrin-A5,-A3, A2 EphB1 Ephrin-B2, -B1, A3 EphB2 Ephrin-B1, -B2, B3 EphB3Ephrin-B1, -B2, B3 EphB4 Ephrin-B2, -B1 EphB5 Unknown EphB6 Unknown

[0020] The work described herein has numerous research and clinicalapplications, which are discussed below.

[0021] As used herein, a transgenic mouse is one which has, incorporatedinto the genome of some or all its nucleated cells, a genetic alterationwhich has been introduced into the mouse or at least one of itsancestors, by the manipulations of man. A transgenic mouse can result,for example, from the introduction of DNA into a fertilized mouse ovumor from the introduction of DNA into embryonic stem cells.

[0022] One embodiment of the present invention is a transgenic mouse,which because of its particular genotype, expresses only in cells ofveins or only in cells of arteries a gene whose RNA transcript orpolypeptide gene product can be detected, for example, by in situhybridization of RNA, by fluorescence, by detection of enzymaticactivity, or by detection of a gene product by antibody binding and adetection system for the bound antibodies.

[0023] A particular embodiment of the present invention is a transgenicmouse of genotype EphrinB2^(+/−), wherein the “minus” allele denotes anallele in which a naturally occurring allele has been deleted, modifiedor replaced with a mutant allele, including a mutant allele which canhave an insertion of an indicator gene. Such a “minus” allele can encodean EphrinB2 ligand which has wild type, altered or no ligand function. Amouse of genotype EphrinB2^(+/tlacZ) has been produced as described inExample 1 and used to demonstrate that arterial endothelial cells andvenous endothelial cells differ genetically from early stages ofdevelopment and that reciprocal interactions, essential for propercapillary bed formation, occur between the two types of vessels. Atransgenic mouse of the same phenotype can be produced by other methodsknown to those of skill in the art. These methods are illustrated belowusing the EphrinB2 gene as an example, but can also be used for anyother vein- or artery-specific gene.

[0024] For example, it is possible to produce a vector carrying aninsertion, a deletion, or one or more point mutations in the EphrinB2gene. The EprhinB2 transgene can be introduced into the genome, via avector carrying a mutagenized EphrinB2 allele, either by introducing thetransgene into a fertilized ovum, by the method of Wagner et al., U.S.Pat. No. 4,873,191 (1989), or by introducing the transgene intoembryonic stem (ES) cells (see, for example, Capecchi, M. R., Science244:1288-1292, 1989), or by other methods.

[0025] An insertion of DNA used to construct a transgenic knockout mousecan have within it a gene whose presence can be readily tested, such asneo, which confers upon its host cells resistance to G418. It is anadvantage of an EphrinB2^(+/−) indicator mouse (e.g.,EphrinB2^(+/taulacZ) to be able to express, under the control of theEphrinB2 promoter, an indicator gene, which can be any gene notendogenously expressed by mice. A particularly advantageous indicatorgene is one which facilitates the detection of EphrinB2 expression,presumably as it is occurring in the wild type allele, by the productionof a gene product that is detectable, for example, by its own lightabsorbance properties, its ability to act upon a substrate to yield acolored product, or its ability to bind to an indicator or dye which isitself detectable.

[0026] Further, alternative methods are available to produce conditionalknockouts or tissue specific knockouts of a gene expressed specificallyin veins or in arteries (i.e., a vein-specific or artery-specific gene),for example by a site-specific recombinase such as Cre (acting at loxPsite) or FLP1 (acting at FRP site) of yeast.

[0027] The bacteriophage P1 Cre-loxP recombination system is capable ofmediating loxP site-specific recombination in both ES cells andtransgenic mice. The site-specific recombinase Cre can also be used in apredefined cell lineage or at a certain stage of development. See, forexample, Gu, H. et al., Science 265:103-106, 1994, in which a DNApolymerase β gene segment was deleted from T cells; see also Tsien, J.Z. et al., Cell 87:1317-1326, 1996, in which Cre/loxP recombination wasrestricted to cells in the mouse forebrain.) The impact of the mutationon these cells can then be analyzed.

[0028] The Cre recombinase catalyzes recombination between 34 bp loxPrecognition sequences (Sauer, B. and Henderson, N., Proc. Natl. Acad.Sci. USA 85:5166-5170, 1988). The loxP sequences can be inserted intothe genome of embryonic stem cells by homologous recombination such thatthey flank one or more exons of a gene of interest (making a “floxed”gene). It is crucial that the insertions do not interfere with normalexpression of the gene. Mice homozygous for the floxed gene aregenerated from these embryonic stem cells by conventional techniques andare crossed to a second mouse that harbors a Cre transgene under thecontrol of a tissue type- or cell type-specific transcriptionalpromoter. In progeny that are homozygous for the floxed gene and thatcarry the Cre transgene, the floxed gene will be deleted by Cre/loxPrecombination, but only in those cell types in which the Cregene-associated promoter is active.

[0029] A gene that encodes a protein which acts to have the effect ofmimicking the phenotype caused by mutations in a vein-specific orartery-specific gene can also be used to achieve the same effect asknockouts in vein-specific or artery-specific genes.

[0030] A mutation in a gene which encodes a product which preventsbinding of ligand to receptor or prevents the functional consequences ofsuch binding and thereby duplicates the phenotype of a vein- orartery-specific gene knockout (e.g., a dominant negative mutant) can beused as an alternative to a knockout. The mutated gene can be put underthe control of a tissue-specific promoter to be expressed in vein orartery, depending on the tissue-specific gene product whose function isto be inhibited.

[0031] In addition, one or more dominant negative alleles of anartery-specific or vein-specific gene can be put under the control of aninducible promoter so that upon induction, the effect of the inhibitionof gene function can be studied. A dominant negative mutant can beisolated or constructed by mutagenesis and methods to make a transgenicmouse.

[0032] Testing to identify the desired mutant or wild type alleles, orfor the identification of other alleles, can be done by PCR on isolatedgenomic DNA, using appropriate primers, or by Southern blots usingappropriate hybridization probes, by a combination of these procedures,or by other methods.

[0033] In addition to the uses of an indicator mouse described in theExamples herein, one use of a mouse having an indicator gene which canmark artery cells is a method for testing an effect of a drug on growthof arteries. The method can comprise administering the drug to a mouse(e.g., embryo, neonate, juvenile, adult, a wound site) having anindicator gene inserted in a gene specifically expressed in arteries,and observing the effect of the drug on the growth of the arteries,compared to the effect in a suitable control mouse having an indicatorgene, not treated with the drug, but maintained under identicalconditions. Similar tests may be performed on an indicator mouse havingan indicator gene which marks vein cells. The effect of the drug can be,for example, to promote growth, to inhibit growth, or to promoteaberrant growth. Administration of the drug can be by any suitable routeknown to those of skill in the art.

[0034] An indicator mouse having an indicator gene inserted in a genespecifically expressed in artery cells can be crossed with a mouse ofanother strain carrying a mutation in a gene which is to be tested forits effect on the growth and development of blood vessels, to allow foreasier visualization of the effects of the mutation specifically onartery cells. In tests similar to those described above, the effect of adrug can be assessed on the mouse which results from this type of cross,to see, for example, whether the effect of the mutation can bealleviated by the drug. In like manner, an indicator mouse having anindicator gene inserted in a gene specifically expressed in vein cellscan be used in a cross with a mouse with a mutation whose effect ongrowth of veins is to be evaluated, and the resulting hybrid used instudies of the growth of veins.

[0035] As a result of the work described herein, it is possible todifferentiate between arterial endothelial cells (arteries) and venousendothelial cells (veins) by taking advantage of the presence of anartery-specific or vein-specific gene product on the surface of thecells. Arterial endothelial cells and venous endothelial cells can eachbe isolated from cells of other tissue types by, for instance, excisionof artery or vein tissue from a sample of mammalian tissue, dissociationof the cells, allowing the cells to bind, under appropriate conditions,to a substance which has some property or characteristic (e.g., amolecule which provides a label or tag, or molecule that has affinityfor both the an artery-specific cell surface protein and another type ofmolecule) that facilitates separation of cells bound to the substancefrom cells not bound to the substance. Separation of the cells can takeadvantage of the properties of the bound substance. For example, thesubstance can be an antibody (antiserum, polyclonal or monoclonal) whichhas been raised against the protein specific to arterial endothelialcells (or to a sufficiently antigenic portion of the protein) andlabeled with a fluorochrome, with biotin, or with another label.Separation of cells bound to the substance can be by FACS, for afluorescent label, by streptavidin affinity column, for a biotin label,by other affinity-based separation methods, or, for example, byantibody-conjugated magnetic beads or solid supports.

[0036] Other means of separation can exploit, for blood vessel cellsbearing an indicator insertion in a gene encoding an artery- orvein-specific protein, the properties of the indicator gene product orportion of fusion protein encoded by the indicator insertion. Forexample, cells producing an artery- or vein-specific fusion protein witha green fluorescent protein portion or a blue fluorescent proteinportion can be separated from non-fluorescent cells by a cell sorter.Cells producing a fusion protein of an artery- or vein-specific proteinportion and an indicator protein or portion with enzymatic or bindingactivity can be detected by the ability of the fusion protein to bind toa fluorescent substrate, for example a substrate for β-galactosidase orβ-lactamase, or to produce a fluorescent product in cells.

[0037] The isolation of arterial endothelial cells and the isolation ofvenous endothelial cells allows for tests of these cell types in cultureto assess the effects of various drugs, growth factors, ligands,cytokines, members of the Eph and Ephrin families of receptors andligands, molecules that bind to cell surface proteins, or othermolecules which can have effects on the growth and development ofarteries and veins. One or more of these substances can be added to theculture medium, and the effects of these additions can be assessed(e.g., by measurements of growth rate or viability, enzyme assays,assays for the presence of cell surface components, incorporation oflabeled precursors into macromolecules such as DNA, RNA or proteins).

[0038] Isolated arterial endothelial cells or isolated venousendothelial cells can be maintained in artificial growth medium and animmortalized cell line can be produced from such isolated cells (i.e.,“transformation”) by infection with one of any number of viruses (e.g.,retroviruses, by transduction of immortalizing oncogene s such as v-mycor SV40 T antigen) known to effectively transform cells in culture. Thevirus can be chosen for its species specificity of infectivity (e.g.,murine ecotropic virus for mouse cells; amphotropic or pseudotypedviruses for human cells). As an alternative to viral transformation,cells can be maintained in culture by propagating the cells in mediumcontaining one or more growth factors.

[0039] Immortalized cell lines derived from either vein or artery cellscan be used to produce cDNA libraries to facilitate study of genesactively expressed in each of these tissues. Further, such cell linescan be used to isolate and identify proteins expressed in the cells, forinstance, by purifying the proteins from conditioned growth medium orfrom the cells themselves.

[0040] As one alternative to using immortalized cell lines of arterialor venous origin, cells or cell lines of non-arterial origin ornon-venous origin (e.g., endothelial cells from other tissues, orfibroblasts) can be genetically altered (by the introduction of one ormore non-endogenously expressed genes) to express an artery-specific orvein-specific cell surface protein, and used in methods to detect andidentify substances that interfere with receptor-ligand interaction.

[0041] Introduction of one or more genes into a cell line can be, forinstance, by transformation, such as by electroporation, by calciumphosphate, DEAE-dextran, or by liposomes, using a vector which has beenconstructed to have an insertion of one or more genes. See Ausubel, F.M. et al, Current Protocols in Molecular Biology, chapter 9, containingsupplements through Supplement 40, Fall, 1997, John Wiley & Sons, NewYork. The introduction of one or more genes to be expressed in a cellline can also be accomplished by viral infection, for example, by aretrovirus. Retroviral gene transfer has been used successfully tointroduce genes into whole cell populations, thereby eliminatingproblems associated with clonal variation.

[0042] The ability to differentiate and to isolate the cells of veinsand arteries allows for a wide variety of applications for a widevariety of purposes. For example, it is now possible to assess theeffects of various agents, such as drugs, diagnostic reagents andenvironmental/dietary factors, on arteries and veins and to determine ifthe effects observed are common to both types of cells or specific toone cell type.

[0043] For example, it can no longer be assumed that angiogenic andanti-angiogenic factors or drugs act equivalently on arterial and venouscells. Isolation and separation of these two cell types, which is madepossible by the present work, allows testing of these angiogenic andanti-angiogenic factors for arterial or venous specificity, which willprovide more selective clinical indications for these drugs. It willalso allow the discovery of new artery- or vein-selective drugs, such asby high-throughput screening of immortalized arterial or venousendothelial cell lines. Existing drugs can also be selectively targetedto arteries or veins by using the proteins described herein as vectors(e.g., viral vehicles having the protein on the viral surface) todeliver drugs (e.g., chemically coupled drugs) to one type of vessel orthe other.

[0044] There are numerous approaches to screening agents for theirselective effects (angiogenic or anti-angiogenic) on arteries and veins.For example, high-throughput screening of compounds or molecules can becarried out to identify angiogenic or anti-angiogenic agents or drugswhich act selectively on arteries or veins or, in some cases, on both.Compounds or molecules which are assessed by such a screening method canbe from a wide variety of sources, such as chemical libraries, cellculture broth or media, cells which have been processed to render theircontents available for screening (plant or animal tissue extracts, forexample) and combinatorial libraries. The compounds or molecules(referred to collectively as agents or drugs) which are screened can bethose already known to have angiogenic or anti-angiogenic activity orthose of unknown effectiveness. In the former case, the screening willbe useful to identify those drugs which act selectively on arterialendothelial cells or on venous endothelial cells and in the latter, itwill be useful to newly identify drugs which have angiogenic oranti-angiogenic activity and to establish the cell type (arterial,venous) on which they act. For example, immortalized cell lines ofarterial or venous origin can be used to screen libraries of compoundsto identify drugs with artery- or vein- specific angiogenic oranti-angiogenic effects. In one embodiment, an assay can be carried outto screen for drugs that specifically inhibit binding of an Ephrinligand to its Eph receptor, such as binding of EphrinB2 to the EphB4receptor, or vice-versa, by inhibition of binding of labeled ligand- orreceptor-Fc fusion proteins to immortalized cells. Alternatively, suchlibraries can be screened to identify members which enhance binding ofan Ephrin ligand to its Eph receptor by enhancing binding of labeledligand- or receptor-Fc fusion proteins to immortalized cells. Drugsidentified through this screening can then be tested in animal models(e.g., models of cancer, arteriovenous malformations or coronary arterydisease) to assess their activity in vivo.

[0045] A drug that inhibits interaction of an artery-specific cellsurface molecule (e.g., an arterial endothelial cell-specific surfacemolecule) with a vein-specific cell surface molecule (e.g., a venousendothelial cell-specific surface molecule) can be identified by amethod in which, for example, the arterial endothelial cell-specificsurface molecule and the veinous endothelial cell-specific surfacemolecule are combined with a drug to be assessed for its ability toinhibit interaction between the cell-specific molecules, underconditions appropriate for interaction between the cell-specificmolecules. The cell-specific molecules may be used in the assay suchthat both are found on intact cells in suspension (e.g., isolatedarterial or venous endothelial cells, immortalized cells derived fromthese, or cells which have been modified to express an artery- orvein-specific cells surface molecule); one cell type is fixed to a solidsupport, and the other molecule specific to the other cell type is insoluble form in a suitable solution; or the molecule specific to onecell type is fixed to a solid support while the molecule specific to theother cell type is found free in a solution that allows for interactionof the cell-specific molecules. Other variations are possible to allowfor the convenient assessment of the interaction between the twodifferent cell-specific molecules.

[0046] In further steps of the assay, the extent to which thecell-specific molecules interact is determined, in the presence of thedrug, and in a separate test (control), in the absence of the drug. Theextent to which interaction of the cell-specific molecules occurs in thepresence and in the absence of the drug to be assessed is compared. Ifthe extent to which interaction of the cell-specific molecules occurs isless in the presence of the drug than in the absence of the drug, thedrug is one which inhibits interaction of the arterial endothelialcell-specific molecule with the venous endothelial cell-specificmolecule. If the extent to which interaction of the cell-specificmolecules occurs is greater in the presence of the drug than in theabsence of the drug, the drug is one which enhances interaction of thearterial endothelial cell-specific molecule with the venous endothelialcell-specific molecule.

[0047] In one embodiment of an assay to identify a substance thatinterferes with interaction of two cell surface molecules, one specificto artery and the other specific to vein (e.g., binding of a ligand to areceptor that recognizes it; interaction between adhesion proteins;interaction between a cell surface protein and a carbohydrate moiety ona cell surface), samples of cells expressing one type of cell surfacemolecule (e.g., cells expressing an Eph receptor, such as a vein-derivedcell line or other cells genetically manipulated to express the Ephreceptor) are contacted with either labeled ligand (e.g., an ephrinligand, a soluble portion thereof, or a soluble fusion protein such as afusion of the extracellular domain and the Fc domain of IgG) or labeledligand plus a test compound or group of test compounds. The amount oflabeled ligand which has bound to the cells is determined. A loweramount of label (where the label can be, for example, a radioactiveisotope, a fluorescent or colormetric label) in the sample contactedwith the test compound(s) is an indication that the test compound(s)interferes with binding. The reciprocal assay using cells expressing aligand (e.g., an Ephrin ligand or a soluble form thereof) can be used totest for a substance that interferes with the binding of a receptor orsoluble portion thereof.

[0048] An assay to identify a substance which interferes withinteraction between artery-specific and vein-specific cell surfaceprotein can be performed with the component (e.g., cells, purifiedprotein, including fusion proteins and portions having binding activity)which is not to be in competition with a test compound, linked to asolid support. The solid support can be any suitable solid phase ormatrix, such as a bead, the wall of a plate or other suitable surface(e.g., a well of a microtiter plate), column pore glass (CPG) or a pinthat can be submerged into a solution, such as in a well. Linkage ofcells or purified protein to the solid support can be either direct orthrough one or more linker molecules.

[0049] Upon the isolation from a mammal of a gene expressing anartery-specific or a vein-specific protein, the gene can be incorporatedinto an expression system for production of a recombinant protein orfusion protein, followed by isolation and testing of the protein invitro. The isolated or purified protein can also be used in furtherstructural studies that allow for the design of agents whichspecifically bind to the protein and can act as agonists or antagonistsof the receptor or ligand activity of the protein.

[0050] In one embodiment, an isolated or purified artery-specific orvein-specific protein can be immobilized on a suitable affinity matrixby standard techniques, such as chemical cross-linking, or via anantibody raised against the isolated or purified protein, and bound to asolid support. The matrix can be packed in a column or other suitablecontainer and is contacted with one or more compounds (e.g., a mixture)to be tested under conditions suitable for binding of the compound tothe protein. For example, a solution containing compounds can be made toflow through the matrix. The matrix can be washed with a suitable washbuffer to remove unbound compounds and non-specifically bound compounds.Compounds which remain bound can be released by a suitable elutionbuffer. For example, a change in the ionic strength or pH of the elutionbuffer can lead to a release of compounds. Alternatively, the elutionbuffer can comprise a release component or components designed todisrupt binding of compounds (e.g., one or more ligands or receptors, asappropriate, or analogs thereof which can disrupt binding orcompetitively inhibit binding of test compound to the protein).

[0051] Fusion proteins comprising all of, or a portion of, anartery-specific or a vein-specific protein linked to a second moiety notoccurring in that protein as found in nature can be prepared for use inanother embodiment of the method. Suitable fusion proteins for thispurpose include those in which the second moiety comprises an affinityligand (e.g., an enzyme, antigen, epitope). The fusion proteins can beproduced by the insertion of a gene specifically expressed in artery orvein cells or a portion thereof into a suitable expression vector, whichencodes an affinity ligand. The expression vector can be introduced intoa suitable host cell for expression. Host cells are disrupted and thecell material, containing fusion protein, can be bound to a suitableaffinity matrix by contacting the cell material with an affinity matrixunder conditions sufficient for binding of the affinity ligand portionof the fusion protein to the affinity matrix.

[0052] In one aspect of this embodiment, a fusion protein can beimmobilized on a suitable affinity matrix under conditions sufficient tobind the affinity ligand portion of the fusion protein to the matrix,and is contacted with one or more compounds (e.g., a mixture) to betested, under conditions suitable for binding of compounds to thereceptor or ligand protein portion of the bound fusion protein. Next,the affinity matrix with bound fusion protein can be washed with asuitable wash buffer to remove unbound compounds and non-specificallybound compounds without significantly disrupting binding of specificallybound compounds. Compounds which remain bound can be released bycontacting the affinity matrix having fusion protein bound thereto witha suitable elution buffer (a compound elution buffer). In this aspect,compound elution buffer can be formulated to permit retention of thefusion protein by the affinity matrix, but can be formulated tointerfere with binding of the compound(s) tested to the receptor orligand protein portion of the fusion protein. For example, a change inthe ionic strength or pH of the elution buffer can lead to release ofcompounds, or the elution buffer can comprise a release component orcomponents designed to disrupt binding of compounds to the receptor orligand protein portion of the fusion protein (e.g., one or more ligandsor receptors or analogs thereof which can disrupt binding of compoundsto the receptor or ligand protein portion of the fusion protein).

[0053] Immobilization can be performed prior to, simultaneous with, orafter contacting the fusion protein with compound, as appropriate.Various permutations of the method are possible, depending upon factorssuch as the compounds tested, the affinity matrix selected, and elutionbuffer formulation. For example, after the wash step, fusion proteinwith compound bound thereto can be eluted from the affinity matrix witha suitable elution buffer (a matrix elution buffer). Where the fusionprotein comprises a cleavable linker, such as a thrombin cleavage site,cleavage from the affinity ligand can release a portion of the fusionwith compound bound thereto. Bound compound can then be released fromthe fusion protein or its cleavage product by an appropriate method,such as extraction.

[0054] One or more compounds can be tested simultaneously according tothe method. Where a mixture of compounds is tested, the compoundsselected by the foregoing processes can be separated (as appropriate)and identified by suitable methods (e.g., PCR, sequencing,chromatography). Large combinatorial libraries of compounds (e.g.,organic compounds, peptides, nucleic acids) produced by combinatorialchemical synthesis or other methods can be tested (see e.g., Ohlmeyer,M. H. J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) andDeWitt, S. H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993),relating to tagged compounds; see also, Rutter, W. J. et al., U.S. Pat.No. 5,010,175; Huebner, V. D. et al., U.S. Pat. No. 5,182,366; andGeysen, H. M., U.S. Pat. No. 4,833,092). Where compounds selected from acombinatorial library by the present method carry unique tags,identification of individual compounds by chromatographic methods ispossible. Where compounds do not carry tags, chromatographic separation,followed by mass spectrophotometry to ascertain structure, can be usedto identify individual compounds selected by the method, for example.

[0055] An in vivo assay useful to identify drugs which act selectivelyon arteries or on veins is also available. It is carried out usingtransgenic animals, such as those described herein, which make itpossible to visualize angiogenic processes. For example, an EphrinB2knockout mouse containing a marker, such as a tau-lacZ insertion, thatmarks all arteries but not veins can be used for a variety of in vivoassays. Other marker genes that can be used, for instance, are genesexpressing alkaline phosphates, blue fluorescent protein or greenfluorescent protein. The mouse, or the targeted allele it contains, canbe used to study angiogenic processes, such as tumor angiogenesis andischemia-associated cardiac neovascularization, in arteries, independentof veins. For example, tumor cells can be implanted in the indicatormouse and arterial vessel growth into the tumor can be visualized bylacZ staining. Alternatively, mice bearing the targeted allele can becrossed with a mouse model of another condition, such as vasculardegeneration or neovascularization, to be visualized. Thearterial-specific aspects of the process can be visually monitored bylacZ staining. An indicator of this type can also be used to assessdrugs for their angiogenic or anti-angiogenic effects.

[0056] A gene product produced specifically by arterial endothelialcells (arteries) and not by other cell types allows for the specifictargeting of drugs, diagnostic agents, tagging labels, histologicalstains or other substances specifically to arteries. In an analogousmanner, a gene product identified as produced specifically by venousendothelial cells (veins) and not detectably produced by other celltypes allows for the specific targeting and delivery of drugs,diagnostic agents, tagging labels, histological stains or othersubstances specifically to veins. The following description of targetingvehicles, targeted agents and methods is presented using EphrinB2 as anillustration of a gene product produced by arterial endothelial cellsand not by vein cells and EphB4 as an illustration of a gene productproduced by venous endothelial cells and not by artery cells. However,this description applies equally well to other artery-specific andvein-specific gene products that can be used to identify these tissuetypes.

[0057] The differential expression of EphrinB2 in arteries and of Eph4in veins allows for the specific targeting of drugs, diagnostic agentsor other substances to the cells of arteries or of veins. A targetingvehicle can be used for the delivery of such a substance. Targetingvehicles which bind specifically to EphrinB2 or to Eph4 can be linked toa substance to be delivered to the cells of arteries or veins,respectively. The linkage can be via one or more covalent bonds, or byhigh affinity non-covalent bonds. A targeting vehicle can be anantibody, for instance, or other compound which binds either to EphrinB2or to EphB4 with high specificity. Another example is an aqueouslysoluble polypeptide having the amino acid sequence of the extracellulardomain of EphB4, or a sufficient portion of the extracellular domain (ora polypeptide having an amino acid sequence conferring a similar enoughconformation to allow specific binding to EphrinB2), which can be usedas a targeting vehicle for delivery of substances to EphrinB2 inarteries. Similarly, a soluble polypeptide having the amino acidsequence of the extracellular domain of EphrinB2 or a sufficientantigenic portion of the extracellular domain (or a polypeptide havingan amino acid sequence conferring a similar enough conformation to allowspecific binding to EphB4), can be used to target substances to EphB4 inveins.

[0058] Targeting vehicles specific to an artery-specific Ephrin ligand(e.g., EphrinB2) or to a vein-specific Eph receptor (e.g., EphB4) havein vivo (e.g., therapeutic and diagnostic) applications. For example, anantibody which specifically binds to EphrinB2 can be conjugated to adrug to be targeted to arteries (e.g., a therapeutic, such as ananti-plaque agent). Alternatively, an antibody which specifically bindsto EphB4 can be used to target a drug to veins. A substance (e.g., aradioactive substance) which can be detected (e.g., a label) in vivo canalso be linked to a targeting vehicle which specifically binds to anartery-specific Ephrin ligand (e.g., EphrinB2) and the conjugate can beused as a labeling agent to identify arteries. Similarly, a detectablelabel can be linked to a targeting vehicle which specifically binds avein-specific Eph receptor (e.g., EphB4) to identify veins.

[0059] Targeting vehicles specific to EphrinB2 or to EphB4 find furtherapplications in vitro. For example, an EphB4-specific targeting vehicle,such as an antibody (a polyclonal preparation or monoclonal) whichspecifically binds to EphB4, can be linked to a substance which can beused as a stain for a tissue sample (e.g., horseradish peroxidase) toprovide a method for the identification of veins in a sample. Likewise,an antibody which specifically binds to EphrinB2 can be used in theidentification of arteries. For instance, in a biopsied tissue sample,as from a tumor, antibody to EphrinB2 can be used to identify arterytissue and to distinguish it from vein tissue, or to identify malformedarteries.

[0060] To treat malformed, painful or cosmetically undesirable veins, anagent which acts against them (e.g, anti-angiogenic factors) can belinked to an EphB4-specific vehicle for local administration to theveins. For example, anti-angiogenic factors can be injected intovaricose veins.

[0061] Targeted agents directed to either an artery-specific Ephrinfamily ligand (e.g., EphrinB2) or a vein-specific Eph family receptor(e.g., EphB4) can also be used when it is desired to produce an effecton both arteries and veins. For example, limited amounts of targetedagents comprising an anti-angiogenic drug and a targeting vehicle toeither EphrinB2, EphB4, or both, can be administered locally to sites ofangiogenesis, such as sites of tumor formation where it is desired toinhibit the growth of blood vessels, or to areas in which increasedvascularization is desired to enhance growth or establishment of bloodvessels.

[0062] Substances that act as agonists or antagonists of anartery-specific Ephrin family ligand (e.g., EphrinB2) or a vein-specificEph family receptor (e.g., EphB4) can be used as angiogenic oranti-angiogenic agents. Drugs that target these molecules willselectively influence arterial and venous angiogenesis. For example,monoclonal antibodies to EphrinB2 or EphB4 can serve as artery- orvein-specific angiogenic or anti-angiogenic agents. As can be concludedfrom the phenotype shown by the EphrinB2^(tlacZ)/EphrinB2^(tlacZ) mutantmice, antagonists of EphrinB2 or antagonists of EphB4 will inhibitangiogenesis. Agents which are agonists of both EphrinB2 and EphB4 willpromote angiogenesis.

[0063] In another example, soluble agonists which comprise theextracellular domain of an Ephrin family ligand or the extracellulardomain of an Eph family receptor fused to the Fc domain of human IgG canbe produced. For example, an EphB4 or an EphrinB2 hybrid protein inwhich the extracellular domain of the membrane protein is fused to theFc domain of human IgG can be used (Wang, H. U. and D. J. Anderson,Neuron 18:383-396 (1997)). See, for examples of methods Stein, E. etal., Genes and Dev. 12:667-678 (1998), regarding experiments onresponses of cells to clustered Ephrin-B1/Fc fusion proteins. Clusteringof these hybrid molecules with anti-human Fc antibodies generatessoluble agonists: Ephrin-derived “ligand-bodies” for Eph receptors, andconversely, Eph-derived “receptor bodies” for Ephrins. Non-clusteredforms of these hybrid molecules can be used as antagonists.

[0064] A further application of isolated arterial endothelial cells andisolated venous endothelial cells is the genetic alteration of theisolated cells and the administration of these cells, preferablyintravenously, to the host mammal from which the cells were isolated, orinto another compatible host, where the cells can be incorporated into ablood vessel of the appropriate type. In this way, the effects of agenetic defect which is manifested in arteries or in veins can beameliorated. It has been demonstrated that circulating endothelial cellprogenitors can migrate to sites of neovascularization and beincorporated into blood vessels (Asahara et al., Science 275:964-967(1997)).

[0065] The introduction of a gene (an endogenous gene that has beenaltered, or a gene originally isolated from a different organism, forexample) into cells can be accomplished by any of several knowntechniques, for example, by vector mediated gene transfer, as byamphotropic retroviruses, calcium phosphate, or liposome fusion, forexample.

[0066] A gene intended to have an effect on arteries or veins in a hostmammal can be delivered to isolated artery cells or isolated vein cellsby the use of viral vectors comprising one or more nucleic acidsequences encoding the gene of interest. Generally, the nucleic acidsequence has been incorporated into the genome of the viral vector. Invitro, the viral vector containing the nucleic acid sequences encodingthe gene can be contacted with a cell and infection can occur. The cellcan then be used experimentally to study, for example, the effect of thegene on growth of artery or vein cells in vitro or the cells can beimplanted into a patient for therapeutic use. The cells to be altered byintroduction or substitution of a gene can be present in a biologicalsample obtained from the patient and used in the treatment of disease,or can be obtained from cell culture and used to dissect developmentalpathways of arteries and veins in vivo and in vitro systems.

[0067] After contact with the viral vector comprising a nucleic acidsequence encoding the gene of interest, the treated artery or vein cellscan be returned or readministered to a patient according to methodsknown to those practiced in the art. Such a treatment procedure issometimes referred to as ex vivo treatment. Ex vivo gene therapy hasbeen described, for example, in Kasid, et al., Proc. Natl. Acad. Sci.USA 87:473 (1990); Rosenberg, et al., New Engl. J. Med. 323:570 (1990);Williams, et al., Nature 310476 (1984); Dick, et al., Cell 42:71 (1985);Keller, et al., Nature 318:149 (1985) and Anderson, et al., U.S. Pat.No. 5,399,346 (1994).

[0068] Generally, viral vectors which can be used therapeutically andexperimentally are known in the art. Examples include the vectorsdescribed by Srivastava, A., U.S. Pat. No. 5,252,479 (1993); Anderson,W. F., et al., U.S. Pat. No. 5,399,346 (1994); Ausubel et al., “CurrentProtocols in Molecular Biology”, John Wiley & Sons, Inc. (1998).Suitable viral vectors for the delivery of nucleic acids to cellsinclude, for example, replication defective retrovirus, adenovirus,parvovirus (e.g., adeno-associated viruses), and coronavirus. Examplesof retroviruses include avian leukosis-sarcoma, mammalian C-type, B-typeviruses, lentiviruses (Coffin, J. M., “Retroviridae: The Viruses andTheir Replication”, In: Fundamental Virology, Third Edition, B. N.Fields, et al., eds., Lippincott-Raven Publishers, Philadelphia, Pa.,(1996)). The mechanism of infectivity depends upon the viral vector andtarget cell. For example, adenoviral infectivity of HeLa cells occurs bybinding to a viral surface receptor, followed by receptor-mediatedendocytosis and extrachromasomal replication (Horwitz, M. S.,“Adenoviruses” In: Fundamental Virology, Third Edition, B. N. Fields, etal., eds., Lippincott-Raven Publishers, Philadelphia, Pa., (1996)).

[0069] The present invention is illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLES Experimental Procedures

[0070] The following experimental procedures were used in the exampleswhich follow.

[0071] Targeted disruption of the EphrinB2 gene. A 200 base pair probestarting from the ATG of the mouse EphrinB2 gene (Bennett, B. D., etal., Proc. Natl. Acad. Sci. USA 92:1866-1870 (1995)) was used to screena 129SVJ genomic library (Stratagene). Analysis of several overlappingclones revealed that the first exon, including the signal sequence, endsat 131 base pairs after the ATG. Further phage analysis and libraryscreens revealed that the rest of the EphrinB2 gene was located at least7 kb downstream from the first exon. To construct a targeting vector(FIG. 1B), a 3 kb Xbal-NcoI fragment whose 3′ end terminated at the ATGwas used as the 5′ arm. A 5.3 kb Tau-lacZ coding sequence (Mombaerts,P., et al., Cell 87:675-686 (1996)) was fused in frame after the ATG.The PGKneo gene (Ma, Q., et al., Neuron 20:469-482 (1998)) was used toreplace a 2.8 kb intronic sequence 3′ to the first exon. Finally, a 3.2kb downstream EcoRI-EcoRI fragment was used as the 3′ arm. Normal (6 kb)and targeted (9 kb) loci are distinguished by HindIII digestion whenprobed with a 1 kb HindIII-XbaI genomic fragment. Electroporation,selection and blastocyst-injection of AB-1 ES cells were performedessentially as described Ma, Q., et al. (Neuron 20:469-482 (1998)), withthe exception that FIAU-selection was omitted. ES cell targetingefficiency via G418 selection was 1 out of 18 clones. Germlinetransmission of the targeted EphrinB2 locus (FIG. 1C) in heterozygousmales was confirmed by Southern blotting of tail DNA of adult mice,using a 1 kb HindIII-XbaI probe. Subsequent genotyping was done bygenomic PCR. Primers for Neo are 5′-AAGATGGATTGCACGCAGGTTCTC-3′ and (SEQID NO.: 1) 5′-CCTGATGCTCTTCGTCCAGATCAT-3′. (SEQ ID NO.: 2)

[0072] Primers for the replaced intronic fragment are5′-AGGACGGAGGACGTTGCCACTAAC-3′ and (SEQ ID NO.: 3)5′-ACCACCAGTTCCGACGCGAAGGGA-3′. (SEQ ID NO.: 4)

[0073] LacZ, PECAM-1, and histological staining. Embryos and yolk sacswere removed between E7.5 and E10.0, fixed in cold 4%paraformaldehyde/PBS for 10 minutes, rinsed twice with PBS, and stainedfor 1 hour to overnight at 37° C. in X-gal buffer (1.3 mg/ml potassiumferrocyanide, 1 mg/ml potassium ferrocyanide, 0.2% Triton X-100, 1 mMMgCl₂, and 1 mg/ml X-gal in PBS, pH 7.2). LacZ stained embryos werepost-fixed and photographed, or sectioned on a cryostat after embeddingin 15% sucrose and 7.5% gelatin in PBS. Procedures for whole mount orsection staining with anti-PECAM-1 antibody (clone MEC 13.3, Pharmingen)were done essentially as described Ma et al. (Neuron 20:469-482 (1998);Fong et al., Nature 376:66-70 (1995)). Horseradish peroxidase-conjugatedsecondary antibodies were used for all PECAM-1 stainings. LacZ-stainedyolk sacs were sectioned in gelatin and then subjected to hematoxylincounter-staining by standard procedures.

[0074] In situ hybridization. In situ hybridization on frozen sectionswas performed as previously described (Birren et al. Development119:597-610 (1993)). Whole-mount in situ hybridization followed aprotocol by Wilkinson, D. G., (Whole-mount in situ hybridization ofvertebrate embryos. pp. 75-83 In: In Situ Hybridization: A PracticalApproach (ed. D. G. Wilkinson) IRL Press, Oxford :75-83 (1992).Bluescript vectors (Stratagene) containing cDNAs for EphB2/Nuk andEphB4/Myk-1 were generated as described Wang, H. U. and Anderson, D. J.(Neuron 18:383-396 (1997)).

Example 1 Targeted Mutagenesis of EphrinB2 in Mice

[0075] Targeted disruption of the EphrinB2 gene was achieved byhomologous recombination in embryonic stem cells. The targeting strategyinvolved deleting the signal sequence and fusing a tau-lacZ indicatorgene in frame with the initiation codon. The expression pattern ofβ-galactosidase in heterozygous (EphrinB2^(tlacZ/+)) embryos wasindistinguishable from that previously reported for the endogenous gene.(Bennett, B. D. et al. Proc. Natl. Acad. Sci. USA 92: 1866-1870 (1995);Bergemann, A. D. et al. Mol. Cell. Bio. 1995:4921-4929 (1995); Wang, H.U. and Anderson, D. J. Neuron 18:383-396 (1997)). While prominentexpression was detected in the hindbrain and somites, lower levels wereobserved in the aorta and heart as early as E8.25. Expression in theyolk sac was first detected at E8.5. Heterozygous animals appearedphenotypically normal. In homozygous embryos, growth retardation wasevident at E10 and lethality occurred with 100% penetrance around E11.No expression of endogenous EphrinB2 mRNA was detected by in situhybridization, indicating that the mutation is a null. Somite polarity,hindbrain segmentation, and the metameric patterning of neural crestmigration (in which EphrinB2 and related ligands have previously beenimplicated Xu, W. et al. Development 121:4005-4016 (1995); Wang, H. U.and Anderson, D. J. Neuron 18:383-396 (1997); Krull, C. E. et al. Curr.Biol. 7:571-580 (1997); Smith, A. et al. Curr. Biol. 7:561-570 (1997))appeared grossly normal in homozygous mutant embryos.

Example 2 Reciprocal Expression Pattern of EphrinB2 and EphB4 inArteries and Veins

[0076] The enlarged heart observed in dying mutant embryos promptedexamination of the expression of EphrinB2^(tlacZ) in the vascular systemin detail. Expression was consistently observed in arteries but notveins. In the yolk sac, for example, the posterior vessels connected tothe vitelline artery, but not the vitelline vein, expressed the gene, asdetected by lacZ staining. In the trunk, labeling was detected in thedorsal aorta, vitelline artery, umbilical artery and its allantoicvascular plexus, but not the umbilical, anterior and common cardinalveins (the umbilical vein is labeled with anti-PECAM-1 antibody). In thehead, labeling was seen in branches of the internal carotid artery, butnot in those of the anterior cardinal vein. In situ hybridization withEphrinB2 cDNA probes confirmed that the selective expression of tau-lacZin arteries correctly reflected the pattern of expression of theendogenous gene. Examination of the expression of the four EphB familygenes, as well as Eph A4/Sek1, which are receptors for EphrinB2 Gale, N.W. et al., Neuron 17:9-19 (1996) revealed complementary expression ofEphB4 in veins but not arteries, including the vitelline vein and itsbranches in anterior portion of the yolk sac.

Example 3 Vasculogenesis Occurs Normally in EphrinB2 Mutant Embryos

[0077] The formation of the major vessels in the trunk was unaffected bythe lack of EphrinB2, as examined by lacZ and PECAM-1 double staining of9 somite embryos. Expression of EphrinB2-lacZ was seen in the dorsalaorta and vitelline artery, but not the umbilical and posterior cardinalveins. The dorsal aorta, vitelline artery, posterior cardinal andumbilical veins, for example, formed, although some dilation andwrinkling of the vessel wall was observed. Similarly the intersomiticvessels originating from the dorsal aorta formed at this stage. BetweenE8.5 and E9.0, the primitive endocardium appeared only mildly perturbedin mutants, while a pronounced disorganization was apparent at E10. Redblood cells developed and circulated normally up to E9.5 in both themutant yolk sac and embryo proper.

Example 4 Extensive Intercalation of Yolk Sac Arteries and VeinsRevealed by EphrinB2 Expression

[0078] In the yolk sac, the vitelline artery and its capillary networkoccupy the posterior region, and the vitelline vein and its capillariesthe anterior region. At E8.5, a stage at which the primary capillaryplexus has formed but remodeling has not yet occurred, asymmetricexpression of EphrinB2-taulacZ in heterozygous embryos was evident atthe interface between the anterior and posterior regions. Apparentlyhomotypic remodeling of β-galactosidase⁺ arterial capillaries intolarger, branched trunks clearly segregated from venous vessels wasevident between E9.0 and E9.5. At this stage, expression of the receptorEphB4 was clearly visible on the vitelline veins but not arteries. Thusarterial and venous endothelial capillaries are already molecularlydistinct following vasculogenesis and prior to angiogenesis.

[0079] While textbook diagrams (Carlson, B. M Patten's Foundations ofEmbryology (1981)) of the yolk sac capillary plexus depict anon-overlapping boundary between the arterial and venous capillary beds,expression of EphrinB2-taulacZ allowed detection of apreviously-unrecognized extensive intercalation between arteries andveins across the entire anterior-posterior extent of the yolk sac; thiswas observed in the heterozygote, but not in the homozygote.Double-labeling for PECAM and β-galactosidase revealed that theinterface between the arteries and veins occurs between microvesselextensions that bridge larger vessels interdigitating en passant.

Example 5 Disrupted Angiogenesis in the Yolk Sac ofEphrinB2^(tlacZ)/EphrinB2^(tlacZ) Embryos

[0080] Defects in yolk sac angiogenesis was were apparent by E9.0 andobvious at E9.5. There was an apparent block to remodeling at thecapillary plexus stage, for both arterial vessels as revealed byβ-galactosidase staining and venous vessels in the anterior region ofthe sac as revealed by PECAM staining. Thus, disruption of the EphrinB2ligand gene caused both a non-autonomous defect in EphB4receptor-expressing venous cells, and an autonomous defect in thearteries themselves.

[0081] This defect was accompanied by a failure of intercalatingbidirectional growth of arteries and veins across the antero-posteriorextent of the yolk sac, so that an interface between EprhinB2-expressingand non-expressing zones at the midpoint of the sac was apparent.(However, small patches of lacZ expression were occasionally visiblewithin the anterior venous plexus, suggesting that some arterialendothelial cells may have become incorporated into venous capillaries.)These observations imply a close relationship between the remodeling ofthe capillary plexus into larger vessels and the intercalating growth ofthese vessels. The large β-galactosidase⁺ vitelline arteries as well asvitelline veins present at the point of entry to the yolk sac of theembryo-derived vasculature appeared unperturbed in the mutant, however.This is consistent with the observation that the mutation does notaffect formation of the primary trunk vasculature. It also argues thatthe yolk sac phenotype is due to a disruption of intrinsic angiogenesisand is not secondary to a failure of ingrowth of embryo-derived vessels.

[0082] Histological staining (hematoxylin) of sectioned yolk sacsrevealed an accumulation of elongated support cells (mesenchymal cellsor pericytes) in close association with the endothelial vessels at E10and E10.5. In mutant yolk sacs, these support cells appeared morerounded, suggesting a defect in their differentiation. Moreover, incontrast to heterozygous yolk sacs, where vessels of different diametersbegan to appear at E9.5 and vessel diameter increased through E10.5,capillary diameter appeared relatively uniform and did not increase withage in the mutants. At E10.5, arteries appear dilated, as if fusion ofvessels occurred without encapsulation by support cells. The mutantcapillaries also failed to delaminate from the basal endodermal layer.

Example 6 Absence of Internal Carotid Arterial Branches and DefectiveAngiogenesis of Venous Capillaries in the Head of Mutant Embryos

[0083] Similar to the yolk sac phenotype, the capillary bed of the headappeared dilated in the mutant, and apparently arrested at the primaryplexus stage. Staining for β-galactosidase revealed that theanterior-most branches of the internal carotid artery failed to developin the mutant. Unlike the case in the yolk sac, therefore, the malformedcapillary beds must be entirely of venous origin. However the anteriorbranches of the anterior cardinal vein formed although they wereslightly dilated. Taken together, these data indicate that in the head,venous angiogenesis is blocked if the normal interaction with arterialcapillaries is prevented. The angiogenic defects observed in the headand yolk sac are unlikely to be secondary consequences of heart defects(see below), since they are observed starting at E9.0 and the embryonicblood circulation appears normal until E9.5.

Example 7 EphrinB2-dependent Signaling Between Endocardial Cells isRequired for Myocardial Trabeculae Formation

[0084] Examination of ligand and receptor expression in wild-type heartsrevealed expression in the atrium of both EphrinB2 (as detected by lacZstaining) and EphB4 (as detected by in situ hybridization). Expressionof both ligand and receptor was also detected in the ventricle in theendocardial cells lining the trabecular extensions of the myocardium.Double-labeling experiments suggested that the ligand and receptor areexpressed by distinct but partially overlapping cell populations,although the resolution of the method does not permit us to distinguishwhether this overlap reflects co-expression by the same cells, or aclose association of different cells. In any case, expression ofEphrinB2 and EphB4 does not define complementary arterial (ventricular)and venous (atrial) compartments of the heart, unlike the extra-cardiacvasculature.

[0085] Heart defects commenced at E9.5 and were apparent in mutantembryos at E10 both morphologically and by wholemount PECAM-1 staining.Sections revealed an absence of myocardial trabecular extensions,although strands of EphrinB2-expressing endocardial cells were stillvisible. Thus, mutation of the ligand-encoding gene caused anon-autonomous defect in myocardial cells, similar to the effect of amutation in the neuregulin-1 gene. (Meyer, D. and Birchmeier, C, Nature378:386-390 (1995)) Paradoxically, however, in this case the EphB4receptor is expressed not on myocardial cells, as is the case for theneuregulin-1 receptors erbB2 and erbB4 (Lee et al., Nature 378:394-398(1995); Gassmann, et al., Nature 378:390-394 (1995), but rather onendocardial cells. Expression of any of the other receptors for Ephrin Bfamily ligands (Eph B1, B2, B3 and A4) was detected in this tissue. Thissuggests that in the heart, ligand-receptor interactions amongendothelial cells may in turn affect interactions with smooth musclecells.

Example 8 Ephrin B2 is Required for Vascularization of the Neural Tube

[0086] In EphrinB2^(tlacZ)/EphrinB2^(tlacZ) embryos capillary ingrowthinto the neural tube failed to occur. Instead, EphrinB2-expressingendothelial cells remained associated with the exterior surface of thedeveloping spinal cord. Comparison of β-galactosidase to pan-endothelialPECAM-1 and EphB4 expression provided no evidence of a separate, venouscapillary network expressing EphB4 in the CNS at this early stage(E9-E10). Rather, expression of a different EphrinB2 receptor, Eph B2,was seen in the neural tube as previously reported Henkemeyer, et al.,Oncogene 9:1001-1014 (1994), where no gross morphological or patterningdefects were detectable. In this case, therefore, the mutation does notappear to cause a non-autonomous phenotype in receptor-expressing cells,rather only an autonomous effect on ligand-expressing cells.

[0087] EQUIVALENTS

[0088] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

1 4 1 24 DNA Artificial Sequence PCR Primer 1 aagatggatt gcacgcaggt tctc24 2 24 DNA Artificial Sequence PCR Primer 2 cctgatgctc ttcgtccaga tcat24 3 24 DNA Artificial Sequence PCR Primer 3 aggacggagg acgttgccac taac24 4 24 DNA Artificial Sequence PCR Primer 4 accaccagtt ccgacgcgaa ggga24

What is claimed is:
 1. A method for altering angiogenesis in a mammal,comprising administering to the mammal, in a therapeutically effectivequantity, a drug which alters binding or interaction of anartery-specific cell surface molecule with a vein-specific cell surfacemolecule in a therapeutically effective quantity.
 2. The method of claim1 wherein the artery-specific cell surface molecule is anartery-specific ligand or receptor and the vein-specific cell surfacemolecule is a vein-specific receptor or ligand.
 3. The method of claim 1wherein the artery-specific cell surface molecule is an Ephrin familyligand and the vein-specific cell surface molecule is an Eph familyreceptor.
 4. The method of claim 3 wherein angiogenesis is inhibited andthe drug interferes with binding or interaction of the artery-specificEphrin family ligand with the vein-specific Eph family receptor.
 5. Themethod of claim 3 wherein angiogenesis is enhanced and the drug enhancesbinding or interaction of the artery-specific Ephrin family ligand withits vein-specific Eph family receptor.
 6. The method of claim 3 whereinthe drug is an antagonist of the artery-specific Ephrin family ligand oran antagonist of the vein-specific Eph family receptor.
 7. The method ofclaim 3 wherein the artery-specific Ephrin family ligand is EphrinB2 andthe vein-specific Eph family receptor is EphB4.
 8. A method forselectively delivering a drug to arteries in a mammal, comprisingadministering to the mammal a complex comprising: a) the drug and b) acomponent which binds an artery-specific cell surface molecule, underconditions appropriate for the component of (b) to bind theartery-specific cell surface molecule, whereby the drug is delivered toarteries.
 9. The method of claim 8, wherein the artery-specific cellsurface molecule is a ligand or receptor.
 10. The method of claim 8,wherein the artery-specific cell surface molecule is an Ephrin familyligand.
 11. The method of claim 10 wherein the drug is ananti-angiogenic drug and the component of (b) is an antibody specificfor the artery-specific Ephrin family ligand, or a receptor of theartery-specific Ephrin family ligand.
 12. The method of claim 10 whereinthe artery-specific Ephrin family ligand is EphrinB2.
 13. The method ofclaim 12 wherein the drug is an anti-angiogenic drug.
 14. The method ofclaim 12 wherein the drug is an angiogenic drug.
 15. A method forselectively delivering a drug to veins in a mammal, comprisingadministering to the mammal a complex comprising: a) the drug, and b) acomponent which binds a vein-specific cell surface molecule, underconditions appropriate for the component of (b) to bind thevein-specific cell surface molecule, whereby the drug is delivered toveins.
 16. The method of claim 15 wherein the vein-specific cell surfacemolecule is a receptor or ligand.
 17. The method of claim 15, whereinthe vein-specific cell surface molecule is a vein-specific Eph familyreceptor.
 18. The method of claim 17 wherein the drug is ananti-angiogenic drug and the component of (b) is an antibody specificfor the vein-specific Eph family receptor, or a ligand of thevein-specific Eph family receptor.
 19. The method of claim 17 whereinthe vein-specific Eph family receptor is EphB4.
 20. The method of claim19 wherein the drug is an anti-angiogenic drug.
 21. The method of claim19 wherein the drug is an angiogenic drug.
 22. A transgenic mouse havingan indicator gene which is detectably expressed in cells of arteries butnot cells of veins.
 23. The mouse of claim 22 wherein the indicator geneis inserted in an artery-specific Ephrin family ligand gene.
 24. Atransgenic mouse of genotype EphrinB2^(+/−).
 25. A transgenic mouse inwhich EphrinB2 genes comprise an insertion that marks all arteries butnot veins.
 26. A transgenic mouse of genotype EphrinB2^(taulacZ/+). 27.A method for identifying artery cells in a mouse having an indicatorgene inserted in one or more alleles of EphrinB2, comprising staining asection of the mouse with a substance appropriate for detection ofexpression of the indicator gene.
 28. A transgenic mouse having anindicator gene which is expressed in venous endothelial cells but not inarterial endothelial cells.
 29. The mouse of claim 28 wherein theindicator gene is inserted in a vein-specific Eph family receptor gene.30. The transgenic mouse of claim 29 wherein the vein-specific Ephfamily receptor gene encodes EphB4.
 31. A transgenic mouse of genotypeEphB4^(+/−).
 32. A method for identifying vein cells in a mouse havingan indicator gene inserted in one or more alleles of EphB4, comprisingstaining a section of the mouse with a substance appropriate fordetection of expression of the indicator gene.
 33. A method for testingan effect of a drug on growth of arteries, comprising administering thedrug to a mouse having an indicator gene inserted in a gene specificallyexpressed in arteries, observing the effect of the drug, and comparingthe effect to that produced in a suitable control mouse.
 34. A methodfor testing an effect of a drug on growth of veins, comprisingadministering the drug to a mouse having an indicator gene inserted in agene specifically expressed in veins, observing the effect of the drug,and comparing the effect to that produced in a suitable control mouse.35. A method for identifying arterial endothelial cells in a tissuesample, comprising contacting the tissue sample with a molecule whichbinds to EphrinB2, wherein said molecule is linked to a label, anddetecting the label, wherein if label is detected on a cell, the cell isan arterial endothelial cell.
 36. The method of claim 35, wherein saidmolecule is an antibody.
 37. A method for identifying venous endothelialcells in a tissue sample, comprising contacting the tissue sample with amolecule which binds to EphB4, wherein said molecule is linked to alabel, and detecting the label, wherein if label is detected on a cell,the cell is a venous endothelial cell.
 38. The method of claim 37,wherein said molecule is an antibody.
 39. A method for directing asubstance to arteries in a mammal, comprising administering to themammal a complex which comprises the substance linked to a moiety whichbinds EphrinB2.
 40. A method for directing a substance to veins in amammal, comprising administering to the mammal a complex which comprisesthe substance linked to a moiety which binds EphB4.
 41. A method foraltering development of blood vessels in a mammal, comprisingadministering to the mammal a soluble polypeptide comprising theextracellular domain of an artery-specific cell surface protein or asoluble polypeptide comprising the extracellular domain of avein-specific cell surface protein.
 42. The method of claim 41 whereinthe artery-specific cell surface protein is an Ephrin family ligand andthe vein-specific cell surface protein is an Eph family receptor. 43.The method of claim 42 wherein the Ephrin family ligand is EphrinB2 andthe Eph family receptor is EphB4.
 44. A method for identifying a drugthat inhibits interaction of an arterial endothelial cell-specificsurface molecule with a venous endothelial cell-specific surfacemolecule, comprising: a) combining: 1) the arterial endothelialcell-specific surface molecule; 2) the venous endothelial cell-specificsurface molecule; and 3) a drug to be assessed for its ability toinhibit interaction between the molecule of (1) and the molecule of (2),under conditions appropriate for interaction between the molecule of (1)and the molecule of (2); b) determining the extent to which the moleculeof (1) and the molecule of (2) interact; and c) comparing the extentdetermined in (b) with the extent to which interaction of the moleculeof (1) and the molecule of (2) occurs in the absence of the drug to beassessed and under the same conditions appropriate for interaction ofthe molecule of (1) with the molecule of (2); wherein if the extent towhich interaction of the molecule of (1) and the molecule of (2) is lessin the presence of the drug than in the absence of the drug, the drug isone which inhibits interaction of the arterial endothelial cell-specificmolecule of (1) with the venous endothelial cell-specific molecule of(2).
 45. The method of claim 44 wherein the arterial endothelialcell-specific surface molecule is an Ephrin family ligand and the venousendothelial cell-specific surface molecule is an Eph family receptor.46. The method of claim 45 wherein the Ephrin family ligand is EphrinB2and the Eph family receptor is EphB4.
 47. A method for identifying adrug that enhances interaction of an arterial endothelial cell-specificsurface molecule with a venous endothelial cell-specific surfacemolecule, comprising: a) combining: 1) the arterial endothelialcell-specific surface molecule; 2) the venous endothelial cell-specificsurface molecule; and 3) a drug to be assessed for its ability toinhibit interaction between the molecule of (1) and the molecule of (2),under conditions appropriate for interaction between the molecule of (1)and the molecule of (2); b) determining the extent to which the moleculeof (1) and the molecule of (2) interact; and c) comparing the extentdetermined in (b) with the extent to which interaction of the moleculeof (1) and the molecule of (2) occurs in the absence of the drug to beassessed and under the same conditions appropriate for interaction ofthe molecule of (1) with the molecule of (2); wherein if the extent towhich interaction of the molecule of (1) and the molecule of (2) isgreater in the presence of the drug than in the absence of the drug, thedrug is one which enhances interaction of the arterial endothelialcell-specific molecule of (1) with the venous endothelial cell-specificmolecule of (2).
 48. The method of claim 47 wherein the arterialendothelial cell-specific surface molecule is an Ephrin family ligandand the venous endothelial cell-specific surface molecule is an Ephfamily receptor.
 49. The method of claim 48 wherein the Ephrin familyligand is EphrinB2 and the Eph family receptor is EphB4.
 50. A methodfor isolating arterial endothelial cells, comprising dissociating cellsof a tissue sample comprising arterial endothelial cells, contacting thedissociated cells with a substance which binds to a cell-surface proteinexpressed specifically on arterial endothelial cells, and separating thecells which have bound the substance from the cells which have not boundthe substance.
 51. A method for isolating arterial endothelial cells,comprising dissociating cells of a tissue sample comprising arterialendothelial cells, contacting the cells with a substance which binds toa cell-surface protein expressed specifically on arterial endothelialcells, wherein said substance is bound to a solid support, removingcells which do not bind to the substance, and releasing the arterialendothelial cells from the solid support.
 52. A method for isolatingvenous endothelial cells, comprising dissociating cells of a tissuesample comprising venous endothelial cells, contacting the dissociatedcells with a substance which binds to a cell-surface protein expressedspecifically on venous endothelial cells, and separating the cells whichhave bound the substance from cells which have not bound the substance.53. A method for isolating venous endothelial cells, comprisingdissociating cells of a tissue sample comprising venous endothelialcells, contacting the cells of the tissue sample with a substance whichbinds to a cell-surface protein expressed specifically on venousendothelial cells, wherein said substance is bound to a solid support,removing cells which do not bind to the substance, and releasing thevenous endothelial cells from the solid support.
 54. Isolated arterialendothelial cells.
 55. Isolated venous endothelial cells.
 56. A methodfor assessing an effect of one or more drugs on arteries, comprisingadding one or more drugs to isolated arterial endothelial cells, andobserving the cells for the effect.
 57. A method for assessing an effectof one or more drugs on veins, comprising adding one or more drugs toisolated venous endothelial cells, and observing the cells for theeffect.
 58. A cell line derived from arterial endothelial cells.
 59. Acell line derived from venous endothelial cells.
 60. A cell line whichproduces a protein that is detectably produced in arteries, but is notdetectably produced in veins.
 61. A cell line which produces a proteinthat is detectably produced in veins, but is not detectably produced inarteries.
 62. A cDNA library produced from isolated arterial endothelialcells.
 63. A cDNA library produced from isolated venous endothelialcells.
 64. A method for identifying a gene which shows differentialexpression in venous endothelial cells compared to arterial endothelialcells, comprising producing a transgenic mouse having an indicatorinsertion gene in a gene to be tested for differential expression, andobserving expression of the indicator insertion gene, wherein adifference in expression of the indicator insertion gene in venousendothelial cells and arterial endothelial cells indicates a gene whichshows differential expression.
 65. A method for modifying arteries in amammal, comprising genetically altering isolated arterial endothelialcells and introducing the altered cells into the mammal.
 66. A methodfor modifying veins in a mammal, comprising genetically alteringisolated veins endothelial cells and introducing the altered cells intothe mammal.
 67. A method for identifying differentially expressed genescomprising; a) dissociating cells of a tissue sample comprising arterialendothelial cells; b) contacting the dissociated cells with a firstsubstance which binds to a cell-surface protein expressed specificallyon arterial endothelial cells; c) separating the arterial endothelialcells which have bound said first substance from the cells which havenot bound said first substance where the bound cells are arterialendothelial cells; d) dissociating cells of a tissue sample comprisingvenous endothelial cells; e) contacting the dissociated cells with asecond substance which binds to a cell-surface protein expressedspecifically on venous endothelial cells; f) separating the cells whichhave bound said second substance from cells which have not bound saidsecond substance wherein cells which have bound are venous endothelialcells; g) subjecting the separated arterial endothelial cells from step(c) and the venous endothelial cells from step (f) to conditions fordifferential expression; and h) identifying differentially expressedgenes.
 68. A gene identified by the method of claim
 67. 69. The gene ofclaim 68 being expressed by an arterial endothelial cell but not avenous endothelial cell.
 70. The gene of claim 68 being expressed by avenous endothelial cell but not an arterial endothelial cell.
 71. Amethod of identifying a gene product expressed by a gene of claim 67 inarterial endothelial cells comprising isolating said gene product froman arterial endothelial cDNA library.
 72. A method of identifying a geneproduct expressed by a gene of claim 67 in venous endothelial cellscomprising isolating said gene product from a venous endothelial cDNAlibrary.
 73. A molecule which binds to the gene product of claim
 71. 74.A molecule which binds to the gene product of claim
 72. 75. The moleculeof claim 73 wherein said molecule is an antibody.
 76. The molecule ofclaim 73 wherein said molecule is a protein or a fragment thereof. 77.The molecule of claim 73 wherein said molecule is a small molecule. 78.The molecule of claim 74 wherein said molecule is an antibody.
 79. Themolecule of claim 74 wherein said molecule is a protein or a fragmentthereof.
 80. The molecule of claim 74 wherein said molecule is a smallmolecule.
 81. A method for identifying differentially expressed genescomprising: a) dissociating cells of a tissue sample comprising arterialendothelial cells; b) contacting the cells with a first substance whichbinds to a cell-surface protein expressed specifically on arterialendothelial cells, wherein said first substance is bound to a firstsolid support; c) removing cells which do not bind to said firstsubstance; d) releasing the arterial endothelial cells from said firstsolid support; e) dissociating cells of a tissue sample comprisingvenous endothelial cells; f) contacting the cells of the tissue samplewith a second substance which binds to a cell-surface protein expressedspecifically on venous endothelial cells, wherein said second substanceis bound to a second solid support; g) removing cells which do not bindto said second substance; h) releasing the venous endothelial cells fromsaid second solid support; i) subjecting the released arterialendothelial cells and the venous endothelial cells in steps (d) and (h)to conditions for differential expression; and j) identifyingdifferentially expressed genes.
 82. The arterial endothelial cellsseparated by the method of claim
 67. 83. The venous endothelial cellsseparated by the method of claim
 67. 84. The arterial endothelial cellsreleased by the method of claim
 81. 85. The venous endothelial cellsreleased by the method of claim
 81. 86. A cell line derived from thearterial endothelial cells of claim
 82. 87. A cell line derived from thevenous endothelial cells of claim
 83. 88. A cell line derived from thearterial endothelial cells of claim
 84. 89. A cell line derived from thevenous endothelial cells of claim
 85. 90. The cell line of claim 86which produces a protein that is detectably produced in arteries, butnot detectably produced in veins.
 91. The cell line of claim 87 whichproduces a protein that is detectably produced in veins, but notdetectably produced in arteries.
 92. The cell line of claim 88 whichproduces a protein that is detectably produced in arteries, but notdetectably produced in veins.
 93. The cell line of claim 89 whichproduces a protein that is detectably produced in veins, but notdetectably produced in arteries.
 94. A method for assessing an effect ofone or more drugs on arteries comprising isolating the arterialendothelial cells of claim 82, adding one or more drugs to said cellsand observing the cells for effect.
 95. A method for assessing an effectof one or more drugs on veins comprising isolating the venousendothelial cells of claim 83, adding one or more drugs to said cellsand observing the cells for effect.
 96. A method for assessing an effectof one or more drugs on arteries comprising isolating the arterialendothelial cells of claim 84, adding one or more drugs to said cellsand observing the cells for effect.
 97. A method for assessing an effectof one or more drugs on veins comprising isolating the venousendothelial cells of claim 85, adding one or more drugs to said cellsand observing the cells for effect.
 98. A method for identifyingdifferentially expressed genes comprising; a) dissociating cells of atissue sample comprising arterial endothelial cells; b) contacting thedissociated cells with a first substance which binds to an Ephrin familyligand expressed specifically on arterial endothelial cells; c)separating the arterial endothelial cells which have bound said firstsubstance from the cells which have not bound said first substance wherethe bound cells are arterial endothelial cells; d) dissociating cells ofa tissue sample comprising venous endothelial cells; e) contacting thedissociated cells with a second substance which binds to an Eph familyreceptor expressed specifically on venous endothelial cells f)separating the cells which have bound said second substance from cellswhich have not bound said second substance wherein cells which havebound are venous endothelial cells; g) subjecting the separated arterialendothelial cells from step (c) and the venous endothelial cells fromstep (f) to conditions for differential expression; and h) identifyingdifferentially expressed genes.
 99. A method for identifyingdifferentially expressed genes comprising; a) dissociating cells of atissue sample comprising arterial endothelial cells; b) contacting thedissociated cells with a first substance which binds to EphrinB2expressed on arterial endothelial cells; c) separating the arterialendothelial cells which have bound said first substance from the cellswhich have not bound said first substance where the bound cells arearterial endothelial cells; d) dissociating cells of a tissue samplecomprising venous endothelial cells; e) contacting the dissociated cellswith a second substance which binds to EphB4 expressed on venousendothelial cells; f) separating the cells which have bound said secondsubstance from cells which have not bound said second substance whereincells which have bound are venous endothelial cells; g) subjecting theseparated arterial endothelial cells from step (c) and the venousendothelial cells from step (f) to conditions for differentialexpression; and h) identifying differentially expressed genes.
 100. Agene identified by the method of claim
 98. 101. The gene of claim 100being expressed by an arterial endothelial cell but not a venousendothelial cell.
 102. The gene of claim 100 being expressed by a venousendothelial cell but not an arterial endothelial cell.
 103. The arterialendothelial cells separated by the method of claim
 98. 104. The venousendothelial cells separated by the method of claim
 98. 105. The arterialendothelial cells separated by the method of claim
 99. 106. The venousendothelial cells separated by the method of claim
 99. 107. A cell linederived from the arterial endothelial cells of claim
 103. 108. A cellline derived from the venous endothelial cells of claim 104.